The present invention relates to a composition, and method for production of an enzymatically inactive urease antigen and the use thereof for urease immunization which leads to the prevention of ammonia toxicity in humans. More specifically, the present invention sets forth a method of production and use of a non-enzymatic and non-toxic urease antigen which serves to immunize humans and animals thus protecting them from the harmful effects of ammonia production by urea splitting bacteria.
Ammonia and ammonia containing molecules are exceedingly toxic for the living organisms. The synthesis of nucleic acids, which are the building blocks of DNA and RNA, and which carry genetic information from generation to generation is impaired by ammonia. (AM J Physiol 223:1004, 1972).
While nitrogen containing compounds are crucial to life, free ammonia is extremely toxic to living organisms. The precise mechanism by which ammonia exerts these toxic effects is imperfectly understood. Various studies have shown that even small increases in the concentration of ammonia in living organisms lead to a decrease in the life span of cells. (Fed. Proc 31:1178, 1972). Specifically, elevated ammonia concentrations have been shown to reduce the life span of both white and red blood cells in human blood, and blood cell counts drop after urease or ammonia injection. Further, even cells that survive a higher concentration of ammonia are functionally impaired and are often permanently injured. It is believed that ammonia exerts its acute toxicity by interfering with enzyme reactions, causing swelling of mitochondria and rhexis of the nucleus. (Proc Soc Exp Biol Med 139:150, 1972).
Ammonia can also lead to improper and delayed wound healing. All healing wounds tend to be quite alkaline because of alkalosis which develops from the loss of CO.sub.2. The CO.sub.2 forms carbonic acid when combined with water and is lost by diffusion from high tissue concentrations to low air concentrations. This alkalosis of surface wounds markedly enhances ammonia intoxication. Ammonia is found as a constituent in 90% of open granulating wounds caused by the action of bacterial urease. (Surg Gynecol & Obs 178:745, 1973).
A manifestation of ammonia toxicity is seen everyday in hospitals that treat patients with impaired liver function such as cirrhosis of the liver. Liver damage can be produced experimentally in animals by the injection of urease into blood which also contains urea. As will be described, the cirrhotics frequently suffer from ammonia intoxication resulting from an increase in free ammonia levels in the blood. The predominant toxic manifestations of high ammonia levels in humans is encephalopathy. The neurological manifestations that accompany this dysfunction of the central nervous system (or encephalon) are so striking that they overshadow the other toxic effects of ammonia. High concentrations of ammonia lead to decreased mentation and mental confusion, symptoms which are not unlike a dementia. Even higher levels of ammonia in the blood stream leads to stupor, coma and eventually, death.
Humans do not form ammonia from urea in the body (Biochem & Biophys Acta 151:646, 1968) and the small amount of ammonia which is formed and circulates in the blood of normal humans is formed in the kidney from glutamine and not by splitting urea. Large quantities of ammonia are formed in the gastrointestinal tract and the highest concentrations of ammonia in the body are found in the portal vein blood coming from that organ. The ammonia is derived by hydrolysis of urea. The harmful effects of this ammonia formed in the colon must be prevented from exerting toxic effects throughout the body's organ systems. Toward this end, the blood from both the small and large intestines is drained by a special set of veins. These veins comprise the portal system. The portal system takes blood directly from the gastrointestinal tract to the liver; only after purification in the liver does the blood enter the general systemic circulation. The large quantity of ammonia formed each day in the gastrointestinal tract is removed by the liver and does not enter the circulating blood in normal people. Intestinal ammonia is mainly produced in the colon. Walser has shown that about 20% of all the urea in the body is converted each day into ammonia in the colon by bacteria. (J Clin Invest 38:1617, 1959). Urea diffuses into the colon from the circulating blood, where it is usually present in concentrations under 20 mg per 100 ml. The urea in the lumen of the large bowel is instantaneously converted to NH.sub.3, which is absorbed by the mucosa of the colon into the portal vein which transports it to the liver. In the liver, NH.sub.3 combines with citrulline to form arginine. The enzyme arginase hydrolyzes the arginine to urea and ornithine. The latter is transformed to citrulline first by the addition of CO.sub.2, and then by NH.sub.3 to complete the cycle described by Krebs and Henseleit. In some situations such as kidney disease, dehydration, or hemorrhage in the gastrointestinal tract, the blood urea level is considerably higher. This increases the amount of ammonia which is formed to very toxic levels. In cirrhotics, some portal venous blood bypasses the liver through collateral veins. Also, because of impaired hepatic function, all of the ammonia is not detoxified by the liver and converted to urea. The ammonia then enters the circulating blood and often produces ammonia intoxication. It is not uncommon for cirrhotics to die in coma after a hemorrhage in the GI tract from a ruptured varix. Blood in the GI tract significantly raises the urea concentration in the intestines and blood increases the conversion of urea to ammonia. Also, animals whose portal vein blood is surgically diverted into the systemic circulation develop severe ammonia intoxication when blood is introduced into the stomach. If the small bowel contents are not allowed to reach the colon in such animals, the ammonia intoxication is very mild, showing that it is the bacterial action on the digested blood in the colon that produces the ammonia intoxication. The seriousness of the colonic content in producing ammonia intoxication in cirrhotics has lead some clinicians to advocate excluding the colon from the GI tract by surgical ileostomy. Another approach to limit ammonia intoxication has been the sterilization of the bowel by the administration of antibiotics. (Proc Exp Biol Med 88:130, 1955).
Urea itself is non-toxic and is present in all tissues of the body. Urea is eventually excreted by the kidneys and appears in the urine. Therefore, it can be seen that a healthy body is ideally suited to handle high ammonia loads encountered in the colon and to render the ammonia harmless. This ammonia is eventually excreted in the form of urea thereby eliminating the harmful metabolite from the body.
Ammonia toxicity is encountered frequently in the alcoholic cirrhotic, although hepatic encephalopathy is encountered in other diseases that result in increased ammonia levels in the blood. Cirrhosis is a general term that includes destruction of functional liver cells as well as a fibrosis or scarring of the liver in response to the injury. Since cirrhosis is a disease of the liver and the liver is predominantly responsible for clearing ammonia from the blood, it can be easily seen how a liver disease of this nature would result in toxic levels of ammonia in the circulating blood.
As noted, the ammonia load delivered to the liver is derived from the gastrointestinal tract. Gastrointestinal ammonia is derived from two sources. One source is ammonia containing compounds that are ingested in the diet and degraded to urea and ammonia. Such compounds are mostly in the form of protein, usually animal protein such as meats, but also to a lesser extent by the ingestion of certain ammonium salts. Because blood contains protein in high concentrations, bleeding in the gastrointestinal tract also promotes ammonia formation.
Urea, the product previously described as an inert product of ammonia metabolism by the liver, is passively secreted into the colon along with other juices that enter the colon from the intestine. The water that is secreted and reabsorbed from the colon is part of the overall salt and water turnover in the body. There is an average not absorption of three liters of water per day in the colon: the colon secretes approximately eight liters of water and reabsorbs eleven liters. (Postgrad Med J 41:435, 1965). The delivery of this large secreted volume to the colon brings about substantial diffusion of urea to the colon which is carried along with the water. The urea is a passive part of the fluid that traverses into the colon during this stage; `passive` means that no energy consuming process secretes urea into the colon but rather that the urea merely accompanies water into the colon. Once in the colon, the urea delivered in this manner is instantaneoulsy converted into ammonia by bacteria present in the colon. As a consequence, urea is never a constituent of colonic contents. (Clin Sci 33:89, 1967).
It has been estimated that 20% of the total body urea is converted to ammonia each day in the colon. Most of the anaerobic bacteria present in the colon contain the enzyme urease, which is capable of splitting urea into its ammonium and carbon dioxide components. This is a process exactly opposite of that accomplished in the liver. It should be noted that all humans have bacteria colonizing the colon and many of these bacteria have urea splitting properties. Urease occurs naturally only in plant life and is not found in human tissues. In the normal person, ammonia created by these micro-organisms is simply reabsorbed in the colon and redirected into the liver by the portal system. In cirrhotic patients, however, these bacteria create an additional ammonia load on the already compromised liver. Indeed, cirrhotics commonly exhibit gastric bleeding which leads to still more colonic ammonia output.
The current state of the art in treating patients with toxic levels of ammonia in their systems is to try to reduce the degree of ammonia production. Patients are placed on low protein diets thereby reducing the amount of ammonia and ammonium compounds delivered to the intestinal system. Lactulose, a sugar not degraded by intestinal enzymes or absorbed in the intestines is not encountered in the diet of humans. This material when ingested reaches the colon where the bacteria of the colon convert it partly to lactic acid. (Gastronenterol 74:544, 1978). This results in a decrease in the pH of the colonic contents further resulting in the conversion of neutral charged ammonia to the positively ionized ammonium ion. Ionized ammonium does not penetrate the cell membrane readily and, therefore, is not absorbed by the intestinal cell in the lumen of the colon. (Am J Surg 119:595, 170). Patients treated for ammonia toxicity are administered large volumes of fluid so as to increase urinary output and large urea excretion by the kidneys.
Sick patients require protein and cannot be put on a diet of zero protein diet content, even though this would decrease the quantity of ammonia derived from protein metabolites. There is an obligate loss of protein every day that must be replenished by ingested foodstuffs. Most of the loss occurs by conversion of protein to urea in the body. Obligate protein loss comes from use of proteins for sources of energy as well as the normal wear and tear of proteins as they perform their daily functions. There are a number of obligate sources of protein loss, not the least of which is loss from the intestinal tract where the mucosa, or the lining of the intestines, is replaced every two to four days in the normal person. As old and worn out cells are sloughed off into the tract, they are replaced by new cells. If the sloughed cells, which are protein rich, come from the beginning of the gastrointestinal tract, they are digested as food and their valuable amino acids are absorbed and recycled. (Anat Record 100:357, 1948). However, cells lost near the end of the tract are past the digestive part of the system and are not broken down into absorbable components. Their protein is lost in stool and must be replaced.
Therefore, it can be seen that the treatment of a cirrhotic suffering from ammonia toxicity requires a delicate titration between a protein need on one hand and the consequences of ammonia toxicity on the other hand. Anything that could sway the balance towards allowing an increased protein intake or more efficient use of a given protein load would be very helpful in treating the cirrhotic patient.
It can also be seen that there is a clear need for methods of decreasing the splitting of urea in the colon to ammonia which is then reabsorbed and taken by the portal system to the liver. This conversion of urea back to ammonia in the colon and then from ammonia back to urea in the liver represents a "futile cycle". This futile cycle wastes the already compromised function of the liver by making it do the same job many times. Preventing the conversion of urea to ammonia in the colon would help decrease the loss of colonic mucosal cells and their protein from the gastrointestinal tract. It has been shown that the ammonia created in the colon reduces the life span of the colonic mucosal cells. These colonic mucosal cells are susceptible to ammonia concentrations. As cells, they are high in protein and their sloughing into the colon accounts for a significant portion of the obligant protein loss from patients and animals. Therefore, by reducing the splitting of urea to ammonia in the colon, two important functions would aide in the treatment of the cirrhotic patient. First, the liver would be spared the chore of driving the futile cycle between ammonia and urea as described above and secondly, the amount of protein that would have to be ingested in the diet to compensate for the obligate protein loss could be reduced. Overall, this would account for a significant decrease in the deliverance of ammonia to the compromised liver and allow the liver to better control ammonia levels. It would also lead to increased use of ingested protein in farm animals reducing the need for protein in the diet.